Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins

Abstract

Multicellular organisms depend on cell production, cell fate specification, and correct patterning to shape their adult body. In plants, auxin plays a prominent role in the timely coordination of these different cellular processes. A well-studied example is lateral root initiation, in which auxin triggers founder cell specification and cell cycle activation of xylem pole-positioned pericycle cells. Here, we report that the E2Fa transcription factor of Arabidopsis thaliana is an essential component that regulates the asymmetric cell division marking lateral root initiation. Moreover, we demonstrate that E2Fa expression is regulated by the LATERAL ORGAN BOUNDARY DOMAIN18/LATERAL ORGAN BOUNDARY DOMAIN33 (LBD18/LBD33) dimer that is, in turn, regulated by the auxin signaling pathway. LBD18/LBD33 mediates lateral root organogenesis through E2Fa transcriptional activation, whereas E2Fa expression under control of the LBD18 promoter eliminates the need for LBD18. Besides lateral root initiation, vascular patterning is disrupted in E2Fa knockout plants, similarly as it is affected in auxin signaling and lbd mutants, indicating that the transcriptional induction of E2Fa through LBDs represents a general mechanism for auxin-dependent cell cycle activation. Our data illustrate how a conserved mechanism driving cell cycle entry has been adapted evolutionarily to connect auxin signaling with control of processes determining plant architecture.

Figure 1.

E2Fa Is Involved in Lateral Root Initiation. (A) to (C) E2Fa, E2Fb, and E2Fc expression kinetics upon synchronous lateral root initiation in roots of Col-0 (A), slr-1 (B), and in pericycle-sorted cells (C). Data represent mean ± se (n ≥ 2). (D) to (G) E2Fa-GFP protein accumulation upon NAA (5 μM) application after 1 h (D) or 5 h (E) in XPP cells counterstained with PI. E3Fa-GFP is visible before (6 h) (F) and after the first asymmetric division (7 h) (G) marking lateral root initiation. Bar = 50 μm (all photomicrographs are at the same magnification). (H) Quantification of emerged (E), nonemerged (NE), and total (T) lateral root primordium density of e2fa T-DNA insertion lines 8 d after germination. (I) Detailed staging of lateral root stage density of e2fa T-DNA insertion lines 8 d after germination. Data represent mean ± se (n ≥ 12; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; two-sided t test).

Figure 2.

LBD18 Binds the E2Fa Promoter and Is Induced upon Lateral Root Initiation. (A) and (B) Interaction of LBD18 with the E2Fa promoter in yeast (A) and in planta (B) as shown by yeast one-hybrid assay and ChIP, respectively. No association was observed with promoters of the closely related genes E2Fb, E2Fc, and DEL1. Interaction of LBD18 was truly positive when both HIS3 (growth on 3-aminotriazole [+ 3-AT] medium) and LacZ (β-galactosidase [X-gal] positive) expression were induced. (C) Confocal microscope images of ProLBD18:NLS-GFP counterstained with PI during lateral root initiation. Time of NAA treatment (5 μM) is indicated in the top left corner. Bar = 50 μm (all photomicrographs are at the same magnification). (D) ProLBD18:GUS expression during different lateral root development stages: before asymmetric division (left), at stage I (middle), and at stage IV (right) . Bar = 50 μm (all photomicrographs are at the same magnification). (E) Root cross section showing GUS staining in XPP cells. Bar = 50 μm. (F) LBD18 transcript accumulation upon synchronized lateral root initiation. Data are mean ± se (n = 3).

Figure 3.

LBD18 Cooperates with the LBD33 Protein during Lateral Root Development. (A) Protoplast transactivation activity assay with a ProE2Fa:fLUC reporter construct, a Pro35S:rLUC normalization construct, and an LBD18 effector construct that is either untagged (Pro35S:LBD18) or fused with VP16 (Pro35S:LBD18-VP16) or SRDX (Pro35S:LBD18-SRDX). Luciferase activity of control cells was arbitrarily set to 1. Data are means ± se (n = 8; *P ≤ 0.05 and ***P ≤ 0.001; two-sided t test). (B) LBD33 expression upon synchronized lateral root initiation. Data are mean ± se (n = 3). (C) Lateral root density of control (Col-0) and lbd33 knockout plants 8 d after germination. Data are mean ± se (n ≥ 12; ***P ≤ 0.001; two-sided t test). (D) Protoplast transactivation activity assay with a ProE2Fa:fLUC reporter construct, a Pro35S:rLUC normalization construct, and LBD18 and/or LBD33 effector constructs. Luciferase activity of control cells was arbitrarily set to 1. Data represent mean ± se (n = 8; **P ≤ 0.01; two-sided t test).

Figure 4.

Confirmation of the LBD18–LBD33 Interaction by FRET Measurements. (A) to (F) Confocal images of transgenic nuclei of Nicotiana benthamiana before ([A] to [C]) and after photobleaching of the acceptor ([D] to [F]). Fluorescence of the fusion proteins LBD33-GFP (donor) and LBD18-mCherry (acceptor) is shown in two channels for the GFP detection (497 to 550 nm) ([A] and [D]), mCherry detection (572 to 636 nm) ([B] and [E]), and merged ([C] and [F]). Bars = 5 μm. (G) FRET efficiency (E_FRET) values in percentages. Donor and acceptor are fusion proteins composed of an LBD protein and the fluorophore GFP and the fluorophore mCherry, respectively. Negative controls are fusions of an LBD protein and GFP alone; corresponding values are considered background noise. Data represent mean ± se (n ≥ 12).

Figure 5.

LBD18 Overexpression Induces E2Fa Expression in Pericycle Cells and Hyperplasia upon Auxin Treatment. (A) E2Fa expression levels in roots of control (Col-0) and Pro35S:LBD18-GFP lines 4.6 and 10.6. Data represent mean ± sd (n = 3). (B) to (E) ProE2Fa:GUS expression in Col-0 ([B] and [C]) versus Pro35S:LBD18-GFP ([D] and [E]) plants. Red and white arrows mark enhanced GUS expression in pericycle cells and pericycle cells flanking lateral root primordia, respectively. Bar = 50 μm (all photomicrographs are at the same magnification). (F) Lateral root density in 8-d-old seedlings that were transferred 4 d after germination to medium supplemented with DMSO or 0.5 μM NAA for an additional 4 d. Data represent mean ± se (n ≥ 12;***P ≤ 0.001; two-sided t test). (G) and (H) Root phenotypes of control (Col-0) (G) and 35S:LBD18-GFP (H) after transfer for 4 d to medium supplemented with 1 μM NAA. Bar = 50 μm (all photomicrographs are at the same magnification). (I) and (J) E2Fa expression in Col-0 (I) and Pro35S:LBD18-GFP (J) after transfer for 4 d to medium supplemented with 1 μM NAA. Bar = 50 μm (all photomicrographs are at the same magnification). (K) Lateral root density in Col-0, lbd18-1, lbd18-2, and both lines complemented with the ProLBD18:E2Fa construct. Data represent mean ± se (n ≥ 12;*** P ≤ 0.001; two-sided t test).

Figure 6.

Aberrant Vascular Tissue Development in E2Fa Knockout Plants. (A) Wild-type venations typically including three or four closed areoles. (B) and (C) E2Fa knockout regularly causing fewer areoles (B) and disconnected veins (C). (D) ProE2Fa:GUS expression pattern in the vascular tissue. Bar = 1 mm (all photomicrographs are at the at same magnification).